Dynamic decreased expression and hypermethylation of secreted frizzled-related protein 1 and 4 over the course of pulmonary fibrosis in mice.

Aberrantly activated Wnt signaling pathway and dysregulation of extracellular antagonists of Wnt signaling have been revealed in pulmonary fibrosis. In this study we evaluated the expression of secreted frizzled-related proteins (SFRPs) and their aberrant promoter methylation to investigate the involvement of epigenetic regulation in pulmonary fibrosis. The pulmonary fibrosis induced by intratracheal injection of bleomycin (BLM) into mice was adopted. The transcription and relative protein expression of SFRPs were detected at Day 7 (D7), D14, and D21. DNA methylation analysis was performed by methylation-specific polymerase chain reaction (MSP). A DNA methyltransferase (DNMT) inhibitor (5-aza-2'-deoxycytidine; 5-aza) was used for demethylation and the relative β-catenin expression levels were measured to assess overactivity of the canonical Wnt signaling pathway. The transcription and protein expression of SFRP1 significantly decreased at D14 and D21, whereas the transcription and protein expression of SFRP4 significantly decreased at D7 and stayed downregulated until D21. The significantly hypermethylated promoters of SFRP1 and SFRP4 resulted in impaired transcription and decreased expression during pulmonary fibrosis in mice. Besides, reactivation of SFRP1 and SFRP4 by 5-aza reduced β-catenin mRNA and protein expression in vivo and in vitro. Animal experiments confirmed that 5-aza could significantly alleviate bleomycin-induced pulmonary fibrosis in mice. Thus, changes of promoter hypermethylation might downregulate SFRP1 and SFRP4 at different stages of pulmonary fibrosis, and the finding supports the usefulness of DNMT inhibitors, which might effectively reverse activation of β-catenin and reduce pulmonary fibrosis in mice. These data provide a possible new direction in the research on pulmonary fibrosis treatments.